Abstract
Purpose :
As age-related macular degeneration (AMD) is marked by mitochondrial dysfunction in the retinal pigment epithelium (RPE), modeling RPE mitochondrial metabolism in vitro may provide insight into AMD pathogenesis. Here, we present the Resipher system for measuring RPE oxygen consumption rates (OCR), a marker for mitochondrial metabolism. We explore the use of Resipher for determining RPE fuel preference and RPE response to AMD-relevant insults. Finally, we employ the device to model RPE hypoxia, an AMD-relevant condition.
Methods :
Utilizing human primary and induced pluripotent stem cell (iPSC) RPE cultures and Resipher, we explore OCR rates before and after certain metabolites or small molecule modulators of metabolism are added to cultures. Additionally, we expose RPE to AMD-relevant insults, including cigarette smoke extract and complement competent serum, measuring the impact of these insults on OCR. Finally, we utilize Resipher to determine culture oxygen availability, monitoring for and modeling hypoxia.
Results :
Resipher allows monitoring for OCR changes before and after an insult or metabolic manipulation, as well as recovery from the manipulation, all over the course of weeks while cells are on standard cell culture plates in a standard cell culture incubator. Utilizing Resipher, we titrate AMD-relevant insults such as cigarette smoke extract so that they cause mild decreases in OCR with weeks-long exposure. We also determine the importance of particular metabolites to RPE metabolism by selectively providing or eliminating that metabolite and seeing the effect on OCR. Finally, we report the impact of media volume on oxygen availability to cultured cells, and the profound effects of media volume on mitochondrial capacity for cells with high oxygen demands, like the RPE. We provide an online calculator for determining if a particular media volume will induce hypoxia for a given cellular OCR. We then model hypoxia, an AMD-relevant insult, simply by adjusting media volume.
Conclusions :
Utilizing Resipher, a novel device for long-term measurement of OCR under standard culture conditions, we have gained insights into RPE fuel preferences, the impact of AMD-relevant insults on mitochondrial capacity, and the impact of media volume on inducing hypoxia. Future studies will determine potential therapeutic agents that can revert RPE mitochondria to a healthier state.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.